The identification of insect species involved in corpses decomposition is of particular importance in estimating the post-mortem interval (PMI) in forensic science, since the PMI is based on information about the life cycle of necrophagous insects. However, the identification of some insect species, especially in their immature stages, may be complicated by many factors, even for experienced taxonomists. Species of the same genus such as Hemilucilia segmentaria (Fabricius) and H. semidiaphana (Rondani) (Diptera: Calliphoridae) are morphologically and behaviorally very similar, but differ in their growth and maturation rates. These species are abundant in forests, exclusively necrophagous and, therefore, are of potential medicolegal importance for estimating the PMI in criminal events that would take place in those areas. In this study, we assessed the usefulness of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in the differentiation of these two species. Two specific regions of mitochondrial DNA, the Cytochrome oxidase subunit I (COI) and the control region (CR), were amplified by PCR and digested using restriction endonucleases. The cleavage patterns generated by the endonucleases DraI and SspI were suitable for differentiating the two Hemilucilia species. This method can be helpful for the forensic entomologist in estimating the PMI because it provides a fast identification, also making possible the use of the insect at any life stage, including immature specimens, regardless of the conditions of preservation (dead or live specimens).345777783